Ethylene is an important gaseous phytohormone regulating plant growth and development in processes such as seed germination, root development, leaf and flower senescence, and fruit ripening, and responds to a variety of stresses (Bleecker, et al. (2000) Annu Rev Cell Dev Biol 16, 1-18; Johnson, et al. (1998) Annu Rev Genet 32, 227-254; Lin, et al. (2009) J Exp Bot 60, 3311-3336; Wang, et al. (2002) Plant Cell 14 Suppl, S131-151). Because of its versatile functions, ethylene has a critical role in adaptation and survival in plants. In the presence of ethylene, etiolated seedlings display a photomorphogenesis phenotype called the triple response: an exaggerated curvature of the apical hook, radial swelling of the hypocotyl, and shortening of the hypocotyl and root (Ecker, J. R. (1995) Science 268, 667-675). The triple response phenotype has been successfully used to identify mutants defective in ethylene biosynthesis or response in Arabidopsis thaliana (Ecker, J. R. (1995) Science 268, 667-675; Chang, et al. (1993) Science 262, 539-544; Guzman, et al. (1990) Plant Cell 2, 513-523; Roman, et al. (1995) Genetics 139, 1393-1409). Further studies of the ethylene mutants revealed the genetic hierarchy of key components in ethylene biosynthesis and signaling transduction in Arabidopsis (Lin, et al. (2009) J Exp Bot 60, 3311-3336; Yoo, et al. (2009) Trends Plant Sci 14, 270-279). Ethylene signaling is initiated by the interaction between the ethylene ligand and its receptors localized in the endoplasmic reticulum (ER) membrane (Chen, et al. (2002) J Biol Chem 277, 19861-19866; Grefen, et al. (2008) Mol Plant 1, 308-320). Binding of ethylene to the receptors inactivates a negative regulator, CTR1, which constitutively represses a positive regulator, EIN2 (Bleecker, et al. (1998) Philos Trans R Soc Lond B Biol Sci 353, 1405-1412). Ethylene receptors activate CTR1 to suppress EIN2 in the absence of ethylene and therefore, function as negative regulators of the ethylene response (Huang, et al. (2003) Plant J 33, 221-233; Qiao, et al. (2009) Genes Dev 23, 512-521). It has been proposed that a functional interaction among the ethylene receptors, CTR1, and EIN2 takes place in or near the ER membrane (Chen, et al. (2002) J Biol Chem 277, 19861-19866; Bisson, et al. (2009) Biochem J424, 1-6; Gao, et al. (2003) J Biol Chem 278, 34725-34732). De-repressed EIN2 stabilizes the otherwise labile transcription factor EIN3 by a yet unknown mechanism (Qiao, et al. (2009) Genes Dev 23, 512-521; Alonso, et al. (1999) Science 284, 2148-2152; Guo, et al. (2003) Cell 115, 667-677; Potuschak, et al. (2003) Cell 115, 679-689). As a consequence, EIN3 activates an array of genes responsible for the ethylene response (Chao, et al. (1997) Cell 89, 1133-1144; Solano, et al. (1998) Genes Dev 12, 3703-3714). Although the ethylene signaling pathway has been elucidated by mainly studying genetic mutants in Arabidopsis, additional factors regulating the key components have been revealed by new approaches (Guo, et al. (2003) Cell 115, 667-677; Potuschak, et al. (2003) Cell 115, 679-689; Yoo, et al. (2008) Nature 451, 789-795), which suggests the use of new methodology to study ethylene function.
Ethylene gas is synthesized in almost all tissues of plants in the presence of oxygen (Yip, et al. (1988) Plant Physiol 88, 553-558). Ethylene biosynthesis involves 3 steps in plants. Methionine is catalyzed to form S-adenosylmethionine (S-AdoMet or SAM) by SAM synthetase. Biosynthesis of ethylene is committed by the conversion of SAM to 1-aminocyclopropane-1-carboxylic acid (ACC) by ACC synthase (ACS) (Yang, et al. (1984) Annual Review of Plant physiology 35, 155-189). ACC is subsequently oxidized to ethylene by ACC oxidase (ACO). Although ACO is constitutively expressed and can be further induced by wounding and ethylene (Barry, et al. (1996) Plant J 9, 525-535; English, et al. (1995) Plant Physiol 109, 1435-1440), the basal activity of ACS is extremely low unless induced by stress signals or at certain developmental stages (Tsuchisaka, et al. (2004) Plant Physiol 136, 2982-3000). Therefore, ACS appears to catalyze the rate-limiting step in ethylene biosynthesis, which is a highly regulated process in higher plant species (Yang, et al. (1984) Annual Review of Plant physiology 35, 155-189). All of the enzymes involved in ethylene biosynthesis, including SAM synthetase, ACC synthase, and ACC oxidase, are encoded by gene families, which suggests a complex and multi-layered regulation of ethylene emanation (Lin, et al. (2009) J Exp Bot 60, 3311-3336).
Genetic mutants defective in the regulation of ethylene biosynthesis have been identified in Arabidopsis (Guzman, et al. (1990) Plant Cell 2, 513-523; Roman, et al. (1995) Genetics 139, 1393-1409). In etiolated seedlings, three ethylene overproduces (eto) mutants, eto1, eto2 and eto3, produce ethylene ranging from 5- to 50-fold higher than that in wild-type Arabidopsis (Guzman, et al. (1990) Plant Cell 2, 513-523; Chae, et al. (2003) Plant Cell 15, 545-559). Arabidopsis ETO2 and ETO3 encode ACS5 and ACS9, two isoforms of type 2 ACS in the gene family (Chae, et al. (2003) Plant Cell 15, 545-559; Vogel, et al. (1998) Proc Natl Acad Sci USA 95, 4766-4771; Yoshida, et al. (2005) BMC Plant Biol 5, 14). ETO1 binds type 2 ACS proteins and interacts with CUL3 in the SCF ubiquitin E3 ligase (Yoshida, et al. (2005) BMC Plant Biol 5, 14; Christians, et al. (2009) Plant J 57, 332-345; Thomann, et al. (2005) FEBS Lett 579, 3239-3245; Wang, et al. (2004) Nature 428, 945-950). ETO1 and ETO1-like (EOL) proteins regulate the protein stability of ETO2/ACS5 and ETO3/ACS9 by the ubiquitin-proteasome pathway (Christians, et al. (2009) Plant J 57, 332-345; Wang, et al. (2004) Nature 428, 945-950). Hypermorphic mutations in eto2-1 and eto3-1 disrupt the protein interactions of ACS5 and ACS9, respectively, with ETO1 resulting in an elevated ACS activity and subsequent ethylene overproduction, which phenocopies the loss-of-function mutations in ETO1 (Guzman, et al. (1990) Plant Cell 2, 513-523; Chae, et al. (2003) Plant Cell 15, 545-559; Vogel, et al. (1998) Proc Natl Acad Sci USA 95, 4766-4771). How the protein-protein interaction between ETO1 and type 2 ACS is regulated by internal and external signals to mediate ethylene production remains largely unclear.
Chemical screening of small molecules as modulators in biological processes of clinically important proteins has been intensively applied in drug discovery (Knight, et al. (2007) Cell 128, 425-430). Small molecules offer advantages of reversible, conditional and rapid effects for functional studies in organisms in which lethality is a critical issue in genetic mutants. In a sense, plant hormones are small molecules that function as bioactive compounds to modulate plant physiology. Chemical genetics has been recently appreciated as a novel methodology to probe plant physiology in Arabidopsis by combining chemical screening and genetics approaches (Blackwell, et al. (2003) Plant Physiol 133, 448-455; Toth, et al. (2010) Trends Plant Sci 15, 81-88).